The use of oligonucleotides for use as probes, primers, linkers, adapters, and antisense agents has been a core element in the field of molecular biology over the past twenty years. Modifications of oligonucleotides have been made to enhance their use as capture and detection probes (for example, the incorporation of biotin, digoxigenen, radioisotopes, fluorescent labels such as fluorescein, reporter molecules such as alkaline phosphatase, etc.). Modifications have also been made to the phosphodiester backbone of nucleic acid molecules to increase their stability. Such modifications involve the use of methyl phosphonates, phosphorothioates, phophorodithioates, 2′-methyl ribose, etc. Other modifications of oligonucleotides have been attempted to increase their cellular uptake or distribution.
A growing class of molecules known as “peptide nucleic acids” (PNAs) resulted from a modification that substituted an amide-linked backbone for the phosphodiester-sugar backbone. One such amide-linked backbone is based on N-(2-aminoethyl)glycine, in which each naturally or non-naturally occurring nucleobase is attached to a N-(2-aminoethyl)glycine unit, and the N-(2-aminoethyl)glycine units are linked together through peptide bonds (see, for example, WO 92/20702; U.S. Pat. No. 5,773,571 issued Jun. 30, 1998 to Nielsen et al. and U.S. Pat. No. 5,539,082 issued Jul. 23, 1996 to Nielsen et al.). The polyamide backbone of PNAs is resistant to both nucleases and proteases.
These nucleic acid analogues can bind both DNA and RNA by Watson-Crick base pairing to form PNA/DNA or PNA/RNA duplexes that have greater thermal stability than corresponding DNA/DNA or DNA/RNA duplexes. Unlike the stability of DNA/DNA or DNA/RNA duplexes, the stability of PNA/DNA or PNA/RNA duplexes is nearly independent of salt concentration. In addition, PNAs bind nucleic acid molecules with greater affinity than DNA or RNA. This is apparent by an 8 to 20 degree drop in melting temperature when a single mismatch is introduced into a PNA/DNA duplex.
An additional feature of PNAs is that homopyrimidine PNAs have been shown to bind complementary DNA or RNA to form (PNA)2/DNA(RNA) triple helices of high thermal stability. Homopyrimidine PNAs can bind to both single-stranded and double-stranded DNA (or RNA). The binding of two single-stranded pyrimidine PNAs to a double-stranded DNA takes place via strand displacement. When PNA strands invade double-stranded DNA, one strand of the DNA is displaced and forms a loop on the side of the (PNA)2/DNA complex area. The other strand of the DNA is locked up in the (PNA)2/DNA triplex structure. The loop area (known as a D loop), being single-stranded, is susceptible to cleavage by enzymes or reagents that can cleave single-stranded DNA.
One drawback of PNAs is their reduced solubility with respect to naturally occurring nucleic acids. Modifications to PNAs to increase their solubility, binding affinity, and specificity have been introduced (see, for example, U.S. Pat. Nos. 5,714,331; 5,736,336; 5,766,855; 5,719,262; 5,786,461; 5,977,296; 6,015,887; and 6,107,470). One such modification is the use of phosphoester bonds in the backbone of nucleic acid analogues, as disclosed by (Efimov, other group). However, these “phosphono PNAs” or “pPNAs” is that they exhibit reduced binding affinity with respect to polyamide or “classical” PNAs.
A common goal in discovery research is identifying genes that are expressed under particular conditions and determining their function. Identification of expressed genes can be used to discover pharmaceutical targets or develop therapeutic strategies. These objectives are often frustrated by the difficulties encountered in isolating RNA transcripts and in obtaining corresponding cDNA clones to particular RNA transcripts that are underrepresented in preparations of messenger RNA and cDNA libraries. Such under-representation can be due to the difficulty in isolating RNA molecules that have short poly A tails or lack poly A tails, or that have secondary structure at their 3′ ends, all of which can confound capture of the RNA molecules by hybridization to oligo T probes. In other cases, the inability to identify a cDNA corresponding to an expressed RNA transcript can be due to the low frequency of cDNA clones corresponding to nonabundant RNAs in cDNA libraries.
There is a need to provide nucleic acid analogues that are stable to nucleases and proteases, that have high binding affinity, binding specificity, and solubility, that are relatively simple to synthesize, and can be used in a variety of applications. In particular, improved methods for the isolation of RNA transcripts and corresponding cDNAs would increase the efficiency of identifying genes that participate in a wide variety of biological functions. The present invention provides these and other benefits.